1. Technical Field
The field of this invention is biological models and therapeutics.
2. Background
Conventionally, gene expression in higher eukaryotes is generally believed to involve the conversion of information encoded in the genome into proteins through the processes of transcription, RNA splicing and export, and translation. Localization of newly synthesized proteins to the correct intracellular compartment, and folding into the correct tertiary structure, are two additional processes at which gene expression could, in principle, be regulated. However, until now the former has been viewed as regulated only with respect to fidelity (Song et al, Cell (2000)100:333-343), degradation (Wiertz et al, Nature (1996) 384:432-438), or signaling to other compartments (Sidrauski et al., Trends Cell Biol (1998) 8:245-249), and not with respect to the information content of the expressed gene. Likewise, folding has also not been viewed at a point of fundamental regulation of gene expression. In general, a dichotomy has been accepted between a protein being either properly folded or misfolded (Ellgaard, et al., Science (1999) 286:1882-1888). The possibility that proteins might have more than one properly folded state and that the cell might be able to select one versus another folded state under particular circumstances or conditions, has not been seriously considered. If either of these possibilities were true, the information content of the genome would be greatly increased. If an invention made it possible to select one conformation versus another, such an invention would be a platform for determining and accessing the information content of the genome in ways that have not been heretofore possible.
In regards to protein folding, a fundamental dogma of modern biology is that primary structure determines secondary structure, which, together with relevant post-translational modifications such as glycosylation, determines the tertiary structure of proteins (Anfinsen, Science (1973) 181:223-230). One revision of this view occurred with the realization that molecular chaperones play a crucial role in enhancing the fidelity of protein folding by preventing inappropriate interactions, thereby facilitating the process of achieving the proper final folded state (Ellis and Hartl, Faseb J (1996) 10:20-26). The recognition that folding is likely initiated in many parts of the molecule at the same time, allowing the chain to funnel towards a minimum energy state without sampling every possibility along the way, constituted a second revision in the generally accepted view of protein folding (Dill and Chan, Nature Struct Biol (1997) 4:10). Neither of these notions considers the possibility that folding might be regulated in the sense of proceeding down one versus another pathway contingent on one versus another set of proteinxe2x80x94protein interactions. If this were the case, protein folding could be amenable to manipulation in ways that could confer diagnostic or therapeutic advantage.
Proteins destined to be secreted from the cell generally contain a signal sequence at the amino terminus that initiates a series of proteinxe2x80x94protein interactions directing the growing chain to the endoplasmic reticulum (ER) membrane and through the translocation channel into the ER lumen (Blobel, PNAS (1980) 77:1496-1500). The roles of signal recognition particle and its receptor (Walter and Johnson, 1996) and of the heterotrimeric Sec 61 complex (Gorlich and Rapoport, Cell (1993) 75:615-630) and of other proteins (Jungnickel and Rapoport, Cell (1996) 82:261-270) in these processes has been elucidated.
Assembly of integral membrane proteins into the membrane of the ER appears to be a complex variation on this general theme, directed by internal signal sequences and stop transfer sequences and hybrid signal-anchor sequences whose interaction with various ER proteins directs the final transmembrane orientation of the polypeptide and often play a subsequent role in anchoring the protein in the bilayer after the chain is released from the translocation channel into the lipid bilayer (Skach and Lingappa, J Biol Chem (1993) 268:23552-23561; Borel and Simon, Cell (1996) 85:379-389; Heinrich et al, Cell (2000) 102:233-244; Moss et al., Mol Biol Cell (1998) 9:2681-2697).
In the case of secretory proteins, the signal sequence is usually cleaved from the growing chain by the ER membrane associated signal peptidase complex, trapping the nascent chain in the ER lumen (Matlack et al., J Cell Biol (1999) 270:6170-6180), with the cleaved signal peptide most likely returned to the cytosol and degraded (Lyko et al., J Biol Chem (1995) 270:19873-19878). In bacteria and primitive eukaryotes such as yeast, a substantial amount of translocation occurs after synthesis is completed (Rapoport et al., J Biol Chem (1999) 380:1143-1150). In such post-translational translocation, a role for the signal sequence as a molecular chaperone to maintain the unfolded state has been proposed (Liu et al., PNAS (1986) 86:9213-9217). However in higher eukaryotes, where most translocation across the ER membrane appears to occur co-translationally, that is, while the chain is still being made, it has generally been assumed that the nascent chain is transferred directly to the ER lumen where folding is initiated (Chen and Helenius, Mol Biol Cell (2000) 11:765-772).
Taken together, the studies presented here suggest the need for several revisions in the current paradigm of protein folding.
First, the simple dichotomy between properly folded and misfolded proteins must be abandoned. It needs to be recognized that protein folding can result in multiple properly folded states which may, potentially, subserve different functions. In most cases these are extremely difficult to detect not only because tools to easily distinguish conformational variants of proteins are limited and not easily applied, but also because the cell complicates the task by degrading variants not wanted at a given point in time.
Second, it must be recognized that the cell has mechanisms by which one folded state (and therefore one function) is chosen (to survive degradative mechanism and be exported to the surface or out of the cell at one time while another folded state (and another function) may be chosen at another time.
Third, it is clear that the machinery and determinants involved in translocation across the ER membrane play an important role in selecting the folding funnel down which a newly synthesized protein proceeds, and that manipulation of either the signal sequence or the machinery with which the chain interacts in the cytosol, membrane or ER lumen are ways to change the folding funnel selected, and therefore, the final conformation or mix of conformations of the protein.
The major implication of the first two revisions of the protein folding paradigm is to increase the information content of the genome enormously. The major implication of the third is to reveal a means of accessing this increased information content of the genome for diagnostic and therapeutic advantage, giving rise to the subject invention.
Besides elucidating these points of background, our studies demonstrate an invention by which the choice of folding funnel can be altered for any given protein in a way that does not change the proteins final primary structure, but which can result in dramatic changes in the protein""s final folded conformation or shape. We demonstrate the signal sequence is able to influence the folding funnel utilized by the growing polypeptide chain, and thereby, to influence the final folded conformation of the resulting newly synthesized protein. Furthermore, cells are able to choose one versus another final conformation through signal sequence-mediated regulation. Because a linear polypeptide sequence folding in different ways could have substantially different shapes, physical properties and biological activities, this new level of regulation greatly increases the information content of the genome and the potential for regulation of gene expression available to the cell.